Cloning the Woolly Mammoth

If you thought reproducing sheep and mice was a leap ahead, you won't believe what the Japanese have in mind.

Look, over here! Hair!" shouts one of the sperm hunters, pointing to a frayed brown tangle protruding from a cliff along the Kolyma River in Siberia. The young man tugs gently at the strands, in the hope that they're attached to a hulk that long ago lay down for the last time in this Ice Age sediment.

Could this, finally, be the hair of a woolly mammoth, its frozen body, and more important, its genitalia, locked inside the crumbling black cliff? Could this be the first step in a bizarre quest led by Japanese biologists to inseminate an Asian elephant with woolly mammoth sperm and selectively breed a fabled prehistoric creature that became extinct thousands of years ago? “I know it sounds unbelievable,” says Kazufumi Goto, “but no science can deny our idea.”

Zimov turns the hair in his hands, rubs it between his fingers and thumb, then poisons the air with his words: “Steppe bison.” He yanks out the rest of the clump to reveal only more loess, no skin or meat.

Many scientists are skeptical of Goto prospects of resurrecting even one woolly mammoth, but few dismiss the plan out of hand—a remarkable sign of how far reproductive technologies have come in only a few years. It’s not the technology that’s stopping Goto, it’s simply a matter of finding a well-preserved woolly mammoth. “The leap of faith is finding the viable sperm or oocytes [cells that become eggs],” says John Critser, scientific director of the Cryobiology Research Institute in Indianapolis. Critser, who has transplanted elephant ovarian tissue into mice and gotten the mice to produce elephant eggs, says that producing an embryo “is not so far-fetched.”

AKIRA IRITANI

If Kazufumi Goto and Akira Iritani come back to Japan with sperm and viable DNA from some other cell of a mammoth, they’ll face a choice: they can try to create either a hybrid or a clone. If they use sperm to create a hybrid, they’ll first need to be sure that the chromosomes are intact. The only way they’ll know is by injecting some of them into mouse eggs. If they move around in the proper pattern, the researchers will shift to elephants. By using only X chromosome–bearing sperm, they’ll ensure that the hybrid will be female. That way they’ll be able to impregnate the female with more mammoth sperm, producing a second hybrid that’s mostly mammoth. But it’s impossible to predict whether a mammoth-elephant hybrid would be fertile. Donkeys and horses produce sterile mules, while swamp buffalo and river buffalo can breed successfully—even though the two buffalo species have a different number of chromosomes.

Cloning a mammoth would be more spectacular than breeding one, but it entails an extra set of hurdles. If the researchers find cells with decent enough DNA, Iritani says, he would inject a mammoth cell’s nucleus into an elephant egg and zap it with electricity to jolt the egg into fertilization mode. But this method demands a cell with a nucleus in good working order, and experts doubt that one could be found in mammoth remains. “I cannot imagine finding a cell in that condition,” says Ian Wilmut, who cloned Dolly. However, he adds, “who is to say what techniques will become available in the future?”

If either method manages to produce a viable embryo, it would be shipped after five cell divisions to a lab in Thailand. Run by Kanok Pavasuthipaisit at Mahidol University, the lab has fertilized Asian elephant eggs in vitro. The embryo would be implanted in a surrogate mother, and 600 days later, if everything went well, a hybrid or pure-blooded mammoth would be born. —R. S

The heyday of the woolly mammoth was the Pleistocene Epoch, stretching from 1.8 million years ago to the end of the last ice age 11,000 years ago. Mammoths thrived particularly well in Siberia, where dry grasslands once stretched for hundreds of miles, supporting a vibrant ecosystem of mammoths, bison, and other jumbo herbivores. They were in turn preyed on by cave lions, wolves, and saber-toothed cats. Famished after the end of the Ice Age by a diet of low-nutrient mosses, and increasingly harried by human hunters, the big grazers dwindled to extinction.

Although most mammoths left behind only their bones, in a few cases the Siberian permafrost preserved mammoth skin and muscle. Most of the cells in this tissue had degraded, but in the past decade scientists managed to rescue a few proteins and fragmented genes to compare with those in living elephants. And until Goto began to think otherwise, that was about as close as anyone thought we’d get to mammoths again.

In the early 1980s, as an assistant professor at Kagoshima University in Japan, Goto applied his nascent understanding of reproductive biology to his country’s pursuit of the ultimate steak. “We have to compete with American beef,” he says. Goto’s task was to use artificial insemination methods to develop beef with more marbling. Getting sperm from well-marbled bulls was easy: he hooked them up to an artificial vagina, a tube warmed with hot water and dabbed with pheromones, and presto. The sperm could then be frozen in glycerol and kept in a freezer indefinitely. At the time, however, collecting cow eggs wasn’t so simple. “I started going to the slaughterhouse,” says Goto. There he could take ovaries from cows with good marbling.

One day in 1986, while peering through a microscope at sperm swarming an egg, Goto had an epiphany. “Once a sperm attaches to the egg’s membrane, it stops moving,” he says. After the two membranes fuse, the sperm is engulfed by the egg and the tail breaks off. “I had been taught that the sperm gets into the oocyte by its own movement,” he says. “I was so surprised to discover that wasn’t true.” It dawned on him that the sperm is already dead by the time it is carried into the egg and delivers its genes.

Intrigued, Goto enlisted a student, Akahiro Kinoshita, to perform a series of experiments using dead sperm. Kinoshita froze and thawed mouse sperm until their membranes were torn up and they lost their metabolism: in other words, they were dead. The genes, on the other hand, were intact. “We could freeze and thaw 20 times, and the sperm DNA would never break.” But when Kinoshita tried injecting the dead sperm into mouse eggs, “it failed thousands of times,” Goto says.

He didn’t give up. He decided mouse eggs were too fragile and moved on to more familiar terrain, instructing his student to try using bull semen and cow eggs. “Finally, one day Kinoshita says, ‘Hey, we got cleavage,’ ” Goto recalls. The egg had begun to divide into an embryo. Skeptical, Goto looked into the microscope. He saw a four-cell cluster where there had been two cells. Kinoshita subsequently implanted the cluster in a cow. “She got pregnant on the first try,” Goto says.

At a 1992 meeting of the International Embryo Transplant Society in Denver, Goto was peppered with question from a reporter about the implications of the discovery. Could viable sperm be recovered from the Ice Man, the 5,000-year-old mummy that had just been discovered in the Alps? Could dead sperm save endangered species? Or extinct ones, such as a mammoth?

“That was the beginning,” says Goto. He wrote to several Russian scientists over the next few years, asking about frozen mammoths. No one wrote back. He queried the Russian embassy in Tokyo, but they asked him paranoid questions: Was he after valuable mammoth tusks?

WOOLLY ANCESTORS

The woolly mammoth was actually a pretty late arrival in elephant evolution. The earliest elephantlike mammals appeared about 50 million years ago and diversified into many different forms that spread over every continent except Australia and Antarctica. The ancestors of mammoths probably split off from the ancestors of today’s African and Asian elephants 5 million years ago. (The mastodons of North America branched off 24 million years ago.) As time passed, the global climate cooled, with ice ages coming and going. Mammoths adapted to the cold, with thick skin, and in many cases a heavy, woolly coat. Mammoths lived in Africa, North America, and Eurasia, reaching 14 feet tall at the shoulder. But huge size wasn’t mandatory for mammoths. When they colonized islands, they often evolved to tiny dimensions—a common pattern for island creatures. On Wrangel Island north of Siberia, they shrank until they were cows, measuring only 6 feet tall at the shoulder. The mammoth fossils on Wrangel are the youngest known in the world. It was there, apparently, that mammoths made their last stand, dying out only 3,800 years ago—700 years after the Egyptians built the great pyramids at Giza. —R. S.

Then, in 1996, Goto met Kazutoshi Kobayashi, an entrepreneur adept at doing business in Russia. In December 1993, two years after the Soviet Union dissolved, Kobayashi flew to Khabarovsk, in eastern Russia, hoping to meet scientists who wanted to license their inventions. In the years since, he has parlayed his Russian connections into a thriving operation that buys technologies such as metal-cutting gas blowtorches and oxygen generators originally designed for the Russian space station Mir.

Kobayashi remembers vividly his first encounter with Goto, in February 1996. “He talked like a child about his dream to make a mammoth, but he was serious,” he says. “My friends always thought I was the most foolish person in the world, but there before me was someone even more foolish. I told Goto we would realize the dream together.”

Kobayashi used his contacts among the government officials that oversee the vast Siberian province Yakutia, to put Goto in contact with Pyotr Lazarev, director of the Mammoth Museum in the capital city, Yakutsk. In August 1996, Lazarev and Goto spent a few days scouting a site near Yakutsk on the Lena River. In early 1997, Kobayashi brought Lazarev to Japan to plan a full-blown expedition for that summer.

Meanwhile, Goto found a scientific partner, Akira Iritani, chairman of the department of genetic engineering at the prestigious Kinki University in Japan. Iritani came with impeccable credentials: in 1986 his lab was one of the first in the world to successfully fertilize rabbit eggs with sperm, a technique now used in humans. Iritani also has an abiding interest in saving endangered species. Kinki University has joined the Frozen Zoo project, in which several U.S. zoos swap samples of frozen semen from wild and zoo animals. Iritani’s lab has sperm on ice from 35 species, including the mountain gorilla and the lion-tailed monkey. “We check on how much damage sperm can bear and still achieve fertilization,” he says. “We hope we will be able to preserve these sperm to maintain a species’ genetic diversity in case a problem with inbreeding develops.”

Goto told Iritani his vision: find mammoth sperm with genes still intact and use it to fertilize a living elephant egg. If the resulting hybrid was a female, he could then fertilize its eggs with more mammoth sperm, breeding a line that would become more and more mammoth. Iritani didn’t want to be limited to the hybrid approach. If mammoth sperm successfully fertilized an elephant egg and the resulting embryo took to a surrogate mother, a half-mammoth/half-elephant wouldn’t be born for 600 days. (Elephants have the longest gestation of any mammal.) A hybrid female should reach reproductive age in 10 to 15 years. It could take 35 years, from start to finish. “I’m 15 years older than Goto,” Iritani says. “I can’t wait 30 years to see a mammoth.”

Web Resources:

Mammoth information from the Royal British Columbia MuseumTheMammoth Saga from the Swedish Museum of Natural HistoryMammuthus